Method and device for microscopy-based imaging of samples

ABSTRACT

A method for performing microscopy-based imaging of samples comprises: loading a sample holder ( 100 ) onto a support ( 50 ) configured to receive the sample holder ( 100 ); moving the sample holder ( 100 ) in a first direction, from a starting position on a first strip of the sample holder ( 100 ), to move the sample holder ( 100 ) relative to an imaging line of a line camera ( 10 ), to capture an image of the first strip of the sample holder ( 100 ); monitoring a focal plane using an autofocus system ( 15 ) as the sample holder ( 100 ) is moved in the first direction; in response to a signal from the autofocus system ( 15 ), moving an objective lens ( 25 ) along the optical axis to adjust the focal plane; and moving the sample holder ( 100 ) in a second direction, to align the imaging line of the line camera ( 10 ) with a position on a second strip of the sample holder ( 100 ).

The present invention relates to a device for microscopy-based analysisof samples. In some examples the device is able to detect the presence,amount, and/or absence of microscopic objects in a sample, such asmicroscopic biological objects.

It is important in various fields to be able to analyse samples quicklyand efficiently and in particular to be able to detect and/or countsmall objects such as bioparticles, molecules, cells, and so on.However, there remains a need to further improve on the capabilities ofmethods and devices in this field.

According to a first aspect, the present invention provides a method forperforming microscopy-based imaging of samples comprising:

loading a sample holder onto a support configured to receive the sampleholder;

moving the sample holder in a first direction, from a starting positionon a first strip of the sample holder, to move the sample holderrelative to an imaging line of a line camera, to capture an image of thefirst strip of the sample holder;

determining (for example, monitoring) a focal plane using an autofocussystem as the sample holder is moved in the first direction;

in response to a signal from the autofocus system, moving an objectivelens along an optical axis to adjust the focal plane (if necessary); and

moving the sample holder in a second direction, to align the imagingline of the line camera with a position on a second strip of the sampleholder.

When the sample holder is loaded, the autofocus system may set aninitial focal plane, before the sample holder is moved.

As the sample holder is moved (for example, to be imaged) the autofocussystem may monitor the focal plane, and may adjust the focal plane asnecessary. Thus, “determining (for example, monitoring) a focal planeusing an autofocus system as the sample holder is moved in the firstdirection; [and] in response to a signal from the autofocus system,moving an objective lens along an optical axis to adjust the focal plane(if necessary)” refers to this monitoring of the focal plane, andadjustment of the focal plane if necessary.

The autofocus system may set the initial focal plane at a surface of thesample holder, and as the sample holder is moved (for example, to beimaged) the autofocus system may monitor the location of the surface ofthe sample holder, and may compensate for any deviations in that surfaceby adjusting the focal plane. If the surface of the sample holder werecompletely optically flat (and perfectly perpendicular to the opticalaxis), no adjustment of the focal plane would be required.

The sample holder may comprise one or a plurality of sample chambers,and the autofocus system may set the initial focal plane at the bottomsurface of the sample chamber(s) in the sample holder. As the sampleholder is moved (for example, to be imaged) the autofocus system maymonitor the location of the bottom surface of the sample chamber(s) inthe sample holder, and may compensate for any deviations in that surfaceby adjusting the focal plane. If the bottom surface of the samplechamber(s) in the sample holder were completely optically flat (andperfectly perpendicular to the optical axis), no adjustment of the focalplane would be required.

Thus, the autofocus system advantageously may be a tracking autofocussystem, such that the autofocus system adjusts the focal plane as thesample holder moves, optionally with a response time sufficiently fastto account for any unevenness in a surface of the sample holder, and inparticular for any unevenness in the bottom surface of the samplechamber(s) in the sample holder.

The focal plane of the line camera may be set at the same plane as thefocal plane determined by the autofocus system. Alternatively, the linecamera may be mounted with a slight offset in the direction of theoptical axis in order to place the focal plane of the line camera at aslightly different level than the focal plane for the autofocus system.

A “strip” is simply the area imaged by the line camera as the sampleholder moves with respect to the imaging line. The strip may have awidth which is defined by the used length W of the imaging line of theline camera (the entire length of the line camera may not be used,depending on the image size produced by the optical system), divided bythe system magnification M, i.e. W/M. Put another way, the strip mayhave a width that is defined by the image size produced by the opticalsystem divided by the system magnification M. The strip may have anotherdimension which is defined by the path which the imaging line takes overthe sample holder, as a result of movement of the sample holder, in onecontinuous motion of the sample holder. So for example, if the sampleholder moves a distance d in a straight line (a linear translationalmovement) the “strip” is a thin rectangular area with width W/M andlength d. If the sample holder moves in a rotational motion (for examplea full rotation of 360°), then the “strip” is an annulus with a width(i.e. the difference between the outer radius of the annulus and theinner radius of the annulus) of W/M.

The optical system may comprise the objective lens and a tube lens.

The optical axis along which the objective lens is moved may beperpendicular to a plane defined by a plane of the sample holder, andfor example may be substantially vertical. The objective lens may bemoved substantially vertically upwardly or downwardly. The support maybe configured to hold the sample holder horizontally, i.e. such that themain planes of the sample holder (its top and bottom surfaces) arehorizontal when the sample holder is held by the support.

Optionally, the line camera images the sample holder from below thesample holder.

In some embodiments, the first and second strips follow radial lines. Inthat case, the image of the first strip may be captured by linearlytranslating the sample holder across the imaging line of the linecamera. That is, the first direction may be a straight line,corresponding to movement inwardly or outwardly along a radial line ofthe sample holder. The imaging line of the line camera may be alignedwith the position on the second strip of the sample holder by rotatingthe sample holder. The second direction may be a rotational direction,e.g. clockwise or anticlockwise, about a vertical axis of the sampleholder.

In some embodiments, the first and second strips follow coaxial circleshaving different radii (i.e. the first and second strips follow at leastpart of the circumference of a circle). In that case, the image of thefirst strip may be captured by rotating the sample holder to move thefirst strip across the imaging line of the line camera. That is, thefirst direction may be a rotational direction, e.g. clockwise oranticlockwise, about a vertical axis of the sample holder. The imagingline of the line camera may be aligned with a position on the secondstrip of the sample holder by linearly translating the sample holder.That is, the second direction may be a straight line, corresponding tomovement inwardly or outwardly along a radial line of the sample holder.

In some embodiments, the first and second strips are parallel. In thatcase, the image of the first strip may be captured by linearlytranslating the sample holder in a first direction across the imagingline of the line camera. That is, the first direction may be a straightline. The imaging line of the line camera may be aligned with the secondstrip of the sample holder by linearly translating the sample holder ina direction at an angle to the first direction, for exampleperpendicular to the first direction. That is, the second direction maybe a straight line at an angle to (for example, perpendicular to) thefirst direction.

The radial line, coaxial circle or parallel lines which the stripsfollow may be thought of as the loci of the centre of the imaging lineof the line camera (i.e. the path traced out by the centre of theimaging line of the line camera).

In any of the foregoing embodiments (where the first and second stripsfollow radial lines, coaxial circles, or parallel lines), the sampleholder optionally is circular in shape. In the cases that the first andsecond strips follow radial lines or coaxial circles, the radial linesmay be radii of the sample holder, and the coaxial circles may beconcentric with the circumference of the sample holder.

Alternatively, the sample holder may have a non-circular shape—forexample, the sample holder may be square or rectangular, or otherwisepolygonal. The “radial” lines in a sample holder having non-circulargeometry are not then radii of the sample holder, but radii of anotional circle on the sample holder.

In each of the foregoing embodiments, wherein the sample holder may becircular in shape, or may have a non-circular shape, and where the firstand second strips follow radial lines, or follow coaxial circles, orfollow parallel lines, the sample holder may comprise a single samplechamber, or a plurality of sample chambers.

Where the sample holder comprises a plurality of sample chambers, afirst plurality of sample chambers may be distributed along the firststrip, and a second plurality of sample chambers may be distributedalong the second strip. In an alternative, a single sample chamber onlymay be located along one of the first or second strips, and a pluralityof sample chambers may be distributed along the other of the second orfirst strip. In another alternative, a single sample chamber only may belocated along the first strip, and a single sample chamber only may belocated along the second strip.

Where a plurality of sample chambers are distributed along a strip orstrips of the sample holder, in some embodiments, the plurality ofsample chambers distributed along a strip of the sample holder are allaligned, so that the radial line, circle, or parallel line which thestrip follows passes through the centre (or a corresponding location) ofeach sample chamber. In some embodiments, the sample chambers generallyfollow the radial line, coaxial circle, or parallel line, but are notall aligned with each other. The sample chambers may for example beshifted relative to one another, for example in an alternating staggeredconfiguration. For example, the centre of some or all of the samplechambers may be offset from the radial line, coaxial circle or parallelline along which they are distributed. For example, a first samplechamber on a radial line, coaxial circle, or parallel line may beshifted to one side of the line, and a neighbouring, second, samplechamber on the line may be shifted to the opposite side of the line. Thenext sample chamber may then be aligned with the first, and the next maybe aligned with the second, etc.

The sample holder may be imaged along a plurality of further strips, inaddition to the first and second strips. These further strips may beimaged in the same manner as the first and second strips are imaged. Thestrips may have the same pattern of sample chambers along each, or theremay be different patterns along each, for example, half of the stripsmay have a first pattern of sample chambers, and half of the strips mayhave a different pattern of sample chambers, or each strip may have adifferent pattern of sample chambers.

The second strip may overlap the first strip, or the first and secondstrips may be spatially separated.

According to a second aspect, the present invention provides a methodfor performing microscopy-based imaging of samples comprising:

loading a sample holder onto a support configured to receive the sampleholder, wherein the sample holder comprises a circular disc withmultiple sample chambers located along each of a plurality of radiallines of the disc;

linearly translating the sample holder, to move the sample holderrelative to an imaging line of a line camera, to capture an image of afirst plurality of sample chambers distributed along a first radialline;

determining (for example, monitoring) a focal plane using an autofocussystem as the sample holder is linearly translated;

in response to a signal from the autofocus system, moving an objectivelens along an optical axis to adjust the focal plane (if necessary); and

rotating the sample holder, to align the imaging line of the line camerawith a position on a second radial line, along which is distributed asecond plurality of sample chambers.

The autofocus system may set an initial focal plane at the bottomsurface of the sample chamber in the sample holder (i.e. the focal planemay be set at the bottom surface of the sample chamber at the imagingposition). As the sample holder is moved to be imaged, the autofocussystem may monitor the location of the bottom surface of the samplechambers in the sample holder, and may compensate for any deviations inthat surface by adjusting the focal plane. If the bottom surface of thesample chambers in the sample holder were completely optically flat (andperfectly perpendicular to the optical axis), no adjustment of the focalplane would be required.

Thus, the autofocus system advantageously may be a tracking autofocussystem, such that the autofocus system adjusts the focal plane as thesample holder moves, optionally with a response time sufficiently fastto account for any unevenness in the bottom surface of the samplechambers in the sample holder.

The focal plane of the line camera may be set at the same plane as thefocal plane determined by the autofocus system. Alternatively, the linecamera may be mounted with a slight offset in the direction of theoptical axis in order to place the focal plane of the line camera at aslightly different level than the focal plane for the autofocus system.

Optionally, the objective lens is moved along an optical axis that isperpendicular to a plane defined by a plane of the sample holder. Forexample, the optical axis may be vertical and the objective lens may bemoved substantially vertically upwardly or downwardly. The support maybe configured to hold the sample holder horizontally, i.e. such that themain planes of the sample holder (its top and bottom surfaces) arehorizontal when the sample holder is held by the support.

Optionally, the line camera images the sample holder from below thesample holder.

According to a third aspect, the present invention provides an imagingdevice for microscopy-based imaging of samples comprising:

a line camera;

a support, configured to receive a sample holder;

an objective lens received by a lens holder, wherein the lens holder isoperable to move the objective lens along an optical axis;

and an autofocus system,

wherein the support is configured to move the sample holder in a firstdirection relative to an imaging line of the line camera to capture animage of a first strip of the sample holder, and wherein the autofocussystem is configured to determine a focal plane, and is configured tooutput a signal which causes the lens holder to translate the objectivelens in order to adjust the focal plane as necessary, during movement ofthe sample holder in the first direction by the support, and wherein thesupport is configured to move the sample holder in a second direction toalign the imaging line of the line camera with a position on a secondstrip of the sample holder.

Optionally, the optical axis along which the objective lens is moved isperpendicular to a plane defined by a plane of the sample holder. Forexample, the optical axis may be vertical, and the objective lens may bemoved substantially vertically upwardly or downwardly. The support maybe configured to hold the sample holder horizontally, i.e. such that themain planes of the sample holder (its top and bottom surfaces) arehorizontal when the sample holder is held by the support.

The imaging device may comprise an optical system comprising theobjective lens and a tube lens.

Optionally, the line camera images the sample holder from below thesample holder.

As noted above, the autofocus system is configured to determine a focalplane, and is configured to output a signal which causes the lens holderto translate the objective lens in order to adjust the focal plane, asnecessary. Optionally, the autofocus system is configured to find theplane of the bottom surface of a sample chamber or a plurality of samplechambers of the sample holder.

The autofocus system may be configured to set an initial focal plane atthe bottom surface of the sample chamber in the sample holder (i.e. thefocal plane may be set at the bottom surface of the sample chamber atthe imaging position). The autofocus system may be configured to monitorthe location of the bottom surface of the sample chamber(s) in thesample holder as the sample holder is moved (for example, to performimaging), and may be configured to compensate for any deviations in thatsurface by outputting a signal for adjusting the focal plane. If thebottom surface of the sample chamber(s) in the sample holder werecompletely optically flat (and perfectly perpendicular to the opticalaxis), no adjustment of the focal plane would be required.

Thus, the autofocus system advantageously may be a tracking autofocussystem, such that the autofocus system is configured to output a signalfor adjusting the focal plane as the sample holder moves, optionallywith a response time sufficiently fast to account for any unevenness ina surface of the sample holder, and in particular for any unevenness inthe bottom surface of the sample chamber(s) in the sample holder.

The focal plane of the line camera may be set at the same plane as thefocal plane determined by the autofocus system. Alternatively, the linecamera may be mounted with a slight offset in the direction of theoptical axis in order to place the focal plane of the line camera at aslightly different level than the focal plane for the autofocus system.

In some embodiments, the first direction is a straight line(corresponding to movement inwardly or outwardly along a radial line ofthe sample holder), and the second direction is a rotational direction(for example, clockwise or anti-clockwise, about a vertical axis of thesample holder). A device configured in this way is particularly suitablefor receiving a sample holder having sample chambers distributed along aplurality of radial lines (i.e. the first and second strips followradial lines), for example, a single sample chamber along each radialline, or a plurality of sample chambers along each radial line.Alternatively, the device is suitable for receiving a sample holderhaving a single sample chamber.

In other embodiments, the first direction is a rotational direction (forexample, clockwise or anti-clockwise, about a vertical axis of thesample holder), and the second direction is a straight line(corresponding to movement inwardly or outwardly along a radial line ofthe sample holder). A device configured in this way is particularlysuitable for receiving a sample holder having sample chambersdistributed along a plurality of coaxial circles, for example, a singlesample chamber along each coaxial circle, or a plurality of samplechambers along each coaxial circle. Alternatively, the device issuitable for receiving a sample holder having a single sample chamber.

In further embodiments, the first direction is a straight line in afirst direction and the second direction is a straight line in adirection at an angle to the first direction, for example perpendicularto the first direction. A device configured in this way is particularlysuitable for receiving a sample holder having sample chambersdistributed along a plurality of parallel lines (i.e. the first andsecond strips follow parallel lines), for example, a single samplechamber along each parallel line, or a plurality of sample chambersalong each parallel line. Alternatively, the device is suitable forreceiving a sample holder having a single sample chamber.

The support may be configured to receive a circular sample holder, or anon-circular sample holder (for example, a square or rectangular sampleholder).

The invention further extends to a system for microscopy-based imagingof samples comprising the imaging device according to the third aspectof the invention, including any or all of theoptional/preferred/advantageous features discussed herein, and a sampleholder, wherein the sample holder may include any or all of theoptional/preferred/advantageous features discussed above in respect ofthe sample holder in the preceding aspects, and/or any or all of theoptional/preferred/advantageous features discussed below.

In a particularly advantageous embodiment, the sample holder comprises aplurality of sample chambers located along each of a plurality of radiallines of the holder.

According to a fourth aspect, the present invention provides an imagingdevice for microscopy-based imaging of samples comprising:

a line camera;

a support, configured to receive a sample holder;

an objective lens received by a lens holder, wherein the lens holder isoperable to translate the objective lens along an axis perpendicular toa plane of the support which receives the sample holder; and

an autofocus system,

wherein the support is configured to translate a received sample holderto move the sample holder relative to the imaging line of the linecamera, such that a first radial line of the sample holder can be imagedby the line camera, and is configured to rotate the received sampleholder, such that a second radial line of the sample holder can beimaged by the line camera, on subsequent translation of the sampleholder,

and wherein the autofocus system is configured to determine a focalplane, and is configured to output a signal which causes the lens holderto translate the objective lens in order to adjust the focal plane,during translation of the sample holder by the support.

The autofocus system may be configured to set the initial focal plane atthe bottom surface of a sample chamber or a plurality of samplechambers(s) in the sample holder. The autofocus system may be configuredto monitor the location of the bottom surface of the sample chamber(s)in the sample holder as the sample holder is moved (for example, toperform imaging), and may be configured to compensate for any deviationsin that surface by outputting a signal for adjusting the focal plane. Ifthe bottom surface of the sample chamber(s) in the sample holder werecompletely optically flat (and perfectly perpendicular to the opticalaxis), no adjustment of the focal plane would be required.

Thus, the autofocus system advantageously may be a tracking autofocussystem, such that the autofocus system is configured to output a signalfor adjusting the focal plane as the sample holder moves, optionallywith a response time sufficiently fast to account for any unevenness ina surface of the sample holder, and in particular for any unevenness inthe bottom surface of the sample chamber(s) in the sample holder.

The focal plane of the line camera may be set at the same plane as thefocal plane determined by the autofocus system. Alternatively, the linecamera may be mounted with a slight offset in the direction of theoptical axis in order to place the focal plane of the line camera at aslightly different level than the focal plane for the autofocus system.

Optionally, the optical axis along which the objective lens istranslated is generally vertical and the lens holder is configured tomove the objective lens substantially vertically upwardly or downwardly.The support may be configured to hold the sample holder horizontally,i.e. such that the main planes of the sample holder (its top and bottomsurfaces) are horizontal when the sample holder is held by the support.

Optionally, the line camera images the sample holder from below thesample holder.

The invention further extends to a system for microscopy-based imagingof samples comprising the imaging device according to the fourth aspectof the invention, including any or all of theoptional/preferred/advantageous features discussed herein, and a sampleholder, wherein the sample holder may include any or all of theoptional/preferred/advantageous features discussed below.

In a particularly advantageous embodiment, the sample holder has acircular shape with one or multiple sample chambers located along eachof a plurality of radial lines of the sample holder.

Embodiments of the invention allow for rapid imaging. Rapid imaging isachieved in part due to the use of a line camera, rather than an areacamera. The use of a line camera enables fast imaging because the sampleholder is constantly moving to capture images along each strip. Thismeans that there is no start-stop motion (except at either end of eachstrip). If an area camera were to be used instead of a line camera, thesample holder would need to be stationary whilst each image is taken,which would lead to a much greater degree of start-stop motion.Start-stop motion is undesirable because the system must be allowed tosettle down after each start and stop. Rapid imaging is advantageous asit allows for imaging of a large area in a relatively short time period.

Advantageously, the line camera does not move. This is advantageous asthe line camera is then not subject to mechanical vibrations, etc.caused by movement, which could impair the quality of the capturedimages.

As noted above, the focal plane may be adjusted by the autofocus systemby moving the objective lens along an axis perpendicular to the plane ofthe sample holder. Moving the objective lens, rather than the sampleholder or line camera, for example, avoids subjecting the sample holderor line camera to rapid, stop-start motions which would necessitate timeto allow the system to settle, and hence slow down the imaging process.

A further advantage of the use of a line camera is that it is readilyadaptable to changes in sample chamber geometry and spacing, i.e. fordifferent designs of sample holder.

Though not limited to such a use, the device and method of the presentinvention are particularly suited to use for antimicrobialsusceptibility testing (AST) analysis. In such an analysis, the growthof pathogens in a sample is determined in a number of differentantimicrobial agents at a number of different concentrations. High speedimaging of a large area allows a large number of conditions (i.e. alarge number of antimicrobial agents at several differentconcentrations) to be tested simultaneously.

The object resolution of the imaging device may be 1 μm or better (i.e.the imaging device may be able to resolve objects with dimensions of 1μm or smaller). Such a device is capable of imaging individualpathogens, for example.

The line camera may comprise a linear digital image sensor, such as aCMOS or CCD image sensor.

The line camera may have a line length defined by the “pixel length” ofeach pixel multiplied by the number of pixels along the line. The linecamera may have a line width defined by the “pixel width” of the pixels.Here, “pixel length” and “pixel width” are labels only; no limitation isimplied on the relative sizes of these dimensions. Thus, the “pixelwidth” may be greater than, equal to, or smaller than the “pixellength”. The “pixel length” dimension is the dimension of the pixels inthe direction that they are aligned end-to-end (side-by-side) to formthe line length. The “pixel width” dimension is the transversedimension. In one embodiment the pixels are square, but this is not arequirement. The pixels may for example be rectangular.

The line camera may have a line width (i.e. pixel width) of 5 μm orless, for example, 4 μm, 3.5 μm, 3 μm, 2.5 μm, or less. The line cameramay have a line width (i.e. pixel width) of 2 μm or more. For example,the line camera has a line width of between 2.5 and 3.5 μm. The valuesdiscussed here (i.e. in the range of 2 μm to 5 μm) are particularlysuitable for use with a system magnification of 10× (but are not limitedto use with such a system magnification); where a different systemmagnification is chosen, the line width may be chosen accordingly.

The selection of line width and numerical aperture is advantageouslymade to achieve a resolution of 1 μm.

The line camera may have a line length (i.e. pixel length×number ofpixels) of 100 mm or less, for example 80 mm or less, 70 mm or less, 60mm or less, or 50 mm or less. The line camera may have a line length(i.e. pixel length×number of pixels) of 10 mm or more, for example 20 mmor more. The line camera may have a line length of between 15 mm and 60mm.

The line camera line length may be selected taking into considerationthe size of the image which will be produced by the objective lens.Thus, for an objective lens which together with the associated tube lensproduces an image having a width of around 20 mm, advantageously a linecamera is selected which has a broadly similar line length (for example,between 20 mm to 30 mm, and in particular, approximately 20 mm).Alternatively, a line camera with a larger line length may be used, butonly a part of this longer line length may be used to image the samplechambers. For example, a line camera with a line length of approximately60 mm may be used, but in practice only approximately 20 mm of the linelength may be utilised for imaging the sample chambers. This is the caseparticularly where the characteristics of the objective lens are suchthat it produces an image having a size which is less than the fullwidth of the imaging camera line width.

The line camera line length is optionally longer than the magnifiedextent of the sample chambers in the direction perpendicular to theradial line, coaxial circle or parallel line along which the samplechambers are distributed. This allows for the entire sample chamber tobe imaged, even in the event of a slight misalignment of the radialline, coaxial circle or parallel line with the imaging line of the linecamera. The magnified extent is the physical extent multiplied by themagnification of the system. (The meaning of the term “extent” isdiscussed in further detail below in the discussion concerning thefeatures of the sample holder.) For example, if the sample chambers havean extent (a physical extent) of 2 mm in the direction perpendicular tothe radial line, coaxial circle or parallel line and the magnificationis 10×, then the magnified extent is 20 mm, and the line camera linelength may be greater than 20 mm, for example, 25 mm.

In other embodiments, the line camera length may be shorter than themagnified extent of the sample chambers in the direction perpendicularto their respective radial line, coaxial circle or parallel line, but inthis case it will not be possible to image the entirety of each samplechamber. In such cases, the line camera length may be selected to imagegreater than 50% of each sample chamber, for example 60%, 70%, 80% or90%.

The line camera imaging rate may be greater than 20 kHz, and for exampleis greater than 40 kHz. For example, the line camera imaging rate may be48 kHz. A faster imaging rate allows the imaging of the sample holder tobe completed more quickly. However, if the line rate is too high, suchthat the exposure time is very short, there may not be sufficientillumination intensity to give an adequate sensor exposure. The linecamera imaging rate may be less than 200 kHz.

The line camera may image a sample in the sample chamber from below thesample holder.

As noted above, the line camera is configured to capture an image. Thisis advantageously an area image, constructed from a plurality ofserially captured line images.

The line camera may output a series of line images (i.e. single lines ofpixels) to a frame grabber which creates a composite image (i.e. thearea image) from the plurality of frames. The imaging device maytherefore comprise a frame grabber.

The composite image may be passed to image analysis software which maycut and truncate the composite image into relevant parts, for example,separate images for each sample chamber, and then analyse the imageareas. The device may comprise a processor for running software toperform the image analysis, and digital storage for storing the softwareand the images.

The imaging device may comprise an illumination source, wherein theillumination source is optionally monochromatic, or a narrow-bandsource. By “narrow-band” source, we mean that(λ_(max)−λ_(min))/λ_(center)<<1, where λ_(max) is the maximumwavelength, λ_(min) is the minimum wavelength, and λ_(center) is thecentral wavelength, of the emission peak of the illumination source. Theuse of such an illumination source (i.e. monochromatic, or a narrow-bandsource) is advantageous as in general it provides for better contrast inthe images. On the other hand, the use of a white or broad-band lightsource would result in chromatic aberration, which would necessitatemodifications to the system to minimize the effect of the chromaticaberration, which is undesirable.

The light source may comprise an incandescent lamp, an arc lamp, an LED,a laser diode, or a laser, for example. Optionally, the light source isan LED, or a plurality of LEDs.

The light from the illumination source may pass through a condenser(comprising a single lens, a compound lens, or a lens set) prior tobeing incident on the sample holder (when held by the support), in orderto shape the light beam emitted from the light source into a shapesuitable for illuminating the sample chamber in the sample holder whichis at the imaging position. The shape of the light beam may be chosentaking into consideration the geometry of the sample chambers in thesample holder. The illumination source and the condenser (where one isprovided) should produce as even illumination as possible over thebottom surface of the sample chamber at the imaging position, andparticularly should produce even illumination at the imaging lineposition (i.e. the position at which the imaging line images the sampleholder).

Advantageously, the illumination homogeneity over an imaged line is 80to 100% of the maximum illumination. Advantageously, the illuminationintensity is within ±10% of the mean intensity over the imaging line.Such illumination homogeneity is advantageous only over the line that isimaged; the illumination may be less homogenous elsewhere.

The illumination wavelength may be chosen taking into consideration thenature of the samples to be imaged, and/or the design capabilities ofthe optical components in the system. In one example, the illuminationwavelength is between 500 and 600 nm, and optionally is in the range of525 to 575 nm. Advantageously, the illumination wavelength is about 550nm. Where the illumination source is a narrow-band source (rather than amonochromatic source), these values may be applicable to the centralwavelength of the source. Such illumination wavelengths are advantageousas the design wavelengths for commercial off-the-shelf optics areusually in this range (i.e. the optics are best corrected for thesewavelength ranges).

The illumination source may comprise a filter configured to pass only aselected wavelength range of the light emitted by the illuminationsource. Such a filter is not necessary if the illumination source ismonochromatic, but may be useful if the illumination source emits aspread of wavelengths. For example, the filter may be a narrow-bandsingle band bandpass filter, with the central wavelength of the band atapproximately the central wavelength of the illumination source. Thebandwidth of the bandpass filter may be less than 200 nm, and may beapproximately 100 nm, or less. The bandpass filter may be placed rightafter the condenser, or between optical elements in the condenser, so asto place the filter in a space with minimum divergence of the rays.

The illumination source may illuminate the sample holder (when held bythe support) from above. That is, the illumination source may beprovided at a position above the support.

The illumination source may comprise a plurality of light sources,and/or a diffuser may be positioned between the illumination source andthe sample holder, for example between a/the condenser and the sampleholder. Such embodiments are particularly advantageous where part of thesample holder (for example, an upper part of the sample holder such as alid) is optically active and has the effect of causing non-uniformity inthe light incident onto the sample chambers. The optically active layermay comprise structures that refract or block light so that theillumination intensity as perceived over the imaged areas in the focalplane is not even, but shows variations dependent on the shape of thelayer above. Such variations may be detrimental to the image andsubsequent image processing. In such a case, the diffuser or pluralityof light sources may act to provide a more even (i.e. more homogeneous)illumination to the sample chambers. Where a plurality of light sourcesis provided, these may be positioned to provide different path lengthsfor illumination of the sample chambers. Where a diffuser is provided,the diffuser may be an optical diffuser which diffuses the light evenly,or it may be an engineered diffuser comprising an engineered surfacehaving structures designed to cancel out the light intensity variationscaused by the optically active part of the sample holder.

The autofocus system may comprise an autofocus system light source,wherein the autofocus light source is an additional light source that isdifferent from the illumination source. The autofocus system lightsource may be a laser.

Optionally, the imaging device comprises a dichroic mirror. Theabove-mentioned bandpass filter may also not be necessary where thedichroic mirror is correctly tuned to provide the proper spectralcharacteristics for the illumination light.

Optionally, the dichroic mirror is broadly transparent to light from theillumination source but reflects the light from the autofocus systemlight source, or is transparent to light from the autofocus system lightsource but broadly reflects the light from the illumination source.

The wavelength of the autofocus system light source may differ from thewavelength of the illumination source. Otherwise, the dichroic mirrorwill not separate the light from the two different sources, in whichcase the function of the autofocus unit may be impaired (for example, asa result of imaging light entering the autofocus unit), and/or lightfrom the autofocus system may be detected by the line camera, which maybe detrimental to imaging.

The light from the autofocus system light source may be configured topass through the objective lens, to be incident onto a bottom surface ofthe sample holder.

The autofocus system advantageously may be a tracking autofocus system,such that the autofocus system adjusts the focal plane as the sampleholder moves, optionally with a response time sufficiently fast toaccount for any unevenness in the sample holder, and in particular forany unevenness in the bottom surface of the sample chambers in thesample holder.

An initial focal plane may be determined by the autofocus system whenthe sample holder is loaded into the device, and then the focal plane isadjusted as the sample holder is moved during imaging. Thus, when thesample holder is loaded into the device, the system is likely out offocus and the autofocus system may perform a search for a surface, andmay find and lock the focal plane to the surface. During imaging (as thesample holder is moved) the autofocus system may monitor the location ofthe surface, and may compensate for any deviations by adjusting thefocal plane.

The autofocus system may be configured to set the focal plane at thebottom surface of the sample chambers in the sample holder, and/or tofollow this surface during imaging.

The focal plane of the line camera may be set at the same plane as thefocal plane determined by the autofocus system. Alternatively, the linecamera may be mounted with a slight offset (for example, greater than 0mm and less than 20 mm, optionally by 15 mm, 10 mm, 5 mm, 2 mm or 1 mm)in the direction of the optical axis in order to place the focal planeof the line camera at a slightly different level than the focal planefor the autofocus system. This may improve imaging of the microscopicobjects in the sample chambers.

Optionally, the autofocus system is configured to adjust the focalposition at least every 1 ms, for example every 0.5 ms, every 0.25 ms,every 0.15 ms, or every 0.1 ms.

Pairing a line camera with an autofocus system that can react fastenough to move the focal position according to unevenness of the sampleholder (in particular, unevenness of the bottom surface of the samplechambers in the sample holder) also leads to more accurate focusing,since only a single line needs to be in focus at a time. In contrast,for an area image, unless the sample holder is completely opticallyflat, parts of the imaged area will be out of focus. With a line camera,a new focus position can be set for each line, if the autofocus systemcan respond quickly enough compared to the line camera line rate. Inpractice, the autofocus system may re-check (and adjust, if necessary)the position of the focal plane every 5 to 10 lines, for example. Toimage a single sample chamber, the line camera may capture thousands oflines (for example, between 10,000 and 15,000), and so the focal planemay be adjusted hundreds or thousands of times, across each samplechamber.

As noted above, the autofocus system is configured to output a signalwhich causes the lens holder to translate the objective lens in order toadjust the focal plane. The lens holder may be able to translate theobjective lens along an axis perpendicular to a plane of the supportwhich receives the sample holder, with a precision of 1 μm or better.Movement of the lens holder may be driven by a linear actuator.

In one configuration, light from the autofocus system light source maypass through the dichroic mirror (for example, to pass through theobjective lens and to be incident onto the bottom surface of the sampleholder), whereas light from the sample holder that has passed throughthe objective lens may be reflected by the dichroic mirror towards theline camera.

In an alternative configuration, light from the autofocus system lightsource is reflected by the dichroic mirror (for example, to pass throughthe objective lens and to be incident onto the bottom surface of thesample holder), whereas light from the sample holder that has passedthrough the objective lens may pass through the dichroic mirror towardsthe line camera.

As noted above, the support is configured to receive the sample holder.Thus, for example, the support may comprise a platform comprising arecessed region shaped to conform to the outer dimensions of the sampleholder, such that, when placed within the recess, the sample holdercannot move laterally.

The platform may be provided on linear tracks attached to the support,and a motor may be provided to drive the platform in either directionalong the tracks. The motor may drive movement of the platform along thetracks via a rack and pinion arrangement, for example.

The support may comprise a through-hole, below the plane at which thesample holder is supported, which allows a portion of the sample holderto be imaged by the line camera, from below.

As further noted above, the support is configured to move a receivedsample holder relative to the imaging line of the line camera, such thata first strip of the sample holder can be imaged by the line camera, andis configured to move the received sample holder, for example such thata second strip of the sample holder can be imaged by the line camera, onsubsequent movement of the sample holder.

The form of the movements of the support may be chosen taking intoconsideration the layout of the sample chambers on the sample holder.

For example, the sample chambers may broadly follow radial lines on thesample holder. Then, in an advantageous example, the support may beconfigured to translate the received sample holder linearly in a firstdirection (for example, from a radially outward position towards thecentre of the sample holder), then to rotate the received sample holderin a second direction (e.g. in a clockwise or anticlockwise direction,about a vertical axis of the sample holder), then to translate thereceived sample holder linearly again in a third direction, the thirddirection being the opposite direction to the first direction (forexample, from a position towards the centre of the sample holder towardsa radially outward position), and then to rotate the received sampleholder again (in the same clockwise or anticlockwise direction). Imagingof the sample chambers may be carried out as the sample holder istranslated in the first direction (for a first radial line of sampleholders) and as the sample holder is translated in the third direction(for a second radial line of sample holders). These steps may berepeated, such that each of the plurality of radial lines of samplechambers on the sample holder is imaged.

In an alternative less advantageous example, the support may beconfigured to translate the received sample holder linearly in a firstdirection (for example, from a radially outward position towards thecentre of the sample holder), then to translate the received sampleholder linearly again in a second direction, the second direction beingthe opposite direction to the first direction (for example, from aposition near the centre of the sample holder towards a radially outwardposition), then to rotate the received sample holder (e.g. in aclockwise or anticlockwise direction). Imaging of the sample chambersmay be carried out as the sample holder is translated in the firstdirection only. These steps may be repeated, such that each of theplurality of radial lines of sample chambers on the sample holder isimaged. This embodiment is less advantageous as the time taken to imagethe sample holder is roughly double that taken in the advantageousembodiment described above.

The sample chambers may broadly follow coaxial circles on the sampleholder (i.e. the sample chambers lie along coaxial circles, each coaxialcircle having a different radius). Then, the support may be configuredto rotate the received sample holder (e.g. in a clockwise oranticlockwise direction, about a vertical axis of the sample holder),then to translate the received sample holder linearly (for example, froma radially outward position towards the centre of the sample holder),then to rotate the received sample holder again. This second rotationmay be in the same direction or the opposite direction to the firstrotation. Imaging of the sample chambers is carried out as the sampleholder is rotated. These steps of rotating and linearly translating maybe repeated, such that each of the plurality of coaxial circles ofsample chambers on the sample holder is imaged.

The sample chambers may broadly follow parallel lines on the sampleholder. Then, the support may be configured to linearly translate thereceived sample holder in a first direction, then to translate thereceived sample holder linearly in a second direction at an angle to thefirst (for example, perpendicular to the first), then to linearlytranslate the received sample holder in a third direction, opposite tothe first direction. Imaging of the sample chambers may be carried outas the sample holder is translated in the first and third direction.These steps of linearly translating may be repeated, such that each ofthe plurality of parallel lines of sample chambers on the sample holderis imaged.

The support may be configured such that it holds the sample holder in afixed position with respect to the vertical axis, i.e. such that thesample holder does not move upwardly or downwardly. The support may beconfigured to hold the sample holder horizontally, i.e. such that themain planes of the sample holder (its top and bottom surfaces) arehorizontal.

The support may comprise a platform lid which is hingedly connected tothe platform. When the platform is translated to an extreme position atone end of the linear tracks, the platform lid may be configured topivot upwardly about the hinged connection, enabling the sample holderto be received by the recessed region. When the platform is moved fromthis extreme position, the platform lid may be configured to pivotdownwardly about the hinged connection, to securely hold the sampleholder.

The sample holder may be loaded onto the support (i.e. into the recessedregion of the platform) at the extreme position. In this position, thesample holder may rest on the recessed region and be prevented fromlateral movement by the recessed region. Optionally, as the platformmoves from the extreme position, the sample holder is further held bythe platform lid, such that the sample holder is prevented from movementupwardly or downwardly by the downward force applied by the platformlid. Movement of the platform lid may be caused by engagement of theplatform lid with a guide rail, which guides the platform lid upwardlyand downwardly, as necessary.

The support may be configured to move the sample holder a distance ofbetween 30 mm and 130 mm, for example 40 mm and 100 mm, for example 50mm to 70 mm, in each linear translation.

The support may be configured to translate the sample holder linearly ata speed of 10 to 20 mm/s, for example 15 mm/s to 18 mm/s. Optionally,the speed of the linear movement may fluctuate to a maximum of ±5%during imaging.

The speed of linear movement of the sample holder may be selected togive an undistorted image, i.e. the speed of linear movement of thesample holder is set taking into consideration the camera imaging rate,the width of the pixel in the line camera imaging line, and themagnification provided by the optical system (comprising the objectivelens and tube lens).

In particular, the speed s of the linear movement of the sample holderis given by:

$s = \frac{{pixel}{width} \times {line}{camera}{imaging}{rate}}{magnification}$

This leads to an undistorted image. Changing the speed and/or camerapixel size and/or pixel aspect ratio and/or system magnification and/orexposure time may distort the image. In some embodiments, a systematicdistortion of the image may be acceptable. For example, if the speed ofthe linear movement of the sample holder is smaller than s, thenmicroscopic objects in the image will be systematically elongated. Ifthe speed of the linear movement of the sample holder is greater than s,then microscopic objects in the image will be systematically contracted.Subsequent image processing may reverse this effect, or may otherwisetake into account this effect.

The support may comprise a drive wheel configured to rotate the sampleholder (for example, about a vertical axis of the sample holder). Forexample, the drive wheel may be located adjacent to the rim of thesample holder, to frictionally engage the rim of the sample holder. Insome embodiments, the drive wheel is pressed to the rim using a springaction. The drive wheel is optionally driven by a second motor, via adrive belt. The support therefore may comprise a second motor and drivebelt. The drive wheel may be configured to disengage from the rim of thesample holder when the platform is translated to an extreme position atone end of the linear tracks. The drive wheel may be configured toengage with the rim of the sample holder when the platform is translatedaway from the extreme position.

The support may be configured to rotate the sample holder (for exampleusing the drive wheel) at a speed of approximately 30° per second.

The support may be configured to align the sample holder in a specificposition such that the starting position for the imaging of the firststrip is known. Optionally, the support comprises a dedicated detector(for example, a photodetector) configured to detect a single alignmentstructure which is present on the sample holder at a distance from thecentre of the sample holder where no other structures are present. Thisalignment structure defines the absolute position, and then apredetermined offset gives the rotational position of the startingimaging position. For example, the device may find the starting positionfor the imaging to within ±500 μm, or within ±100 μm, or even within ±50μm, as measured at the outermost sample chamber.

Optionally, a fine positioning procedure is carried out. The procedurecomprises: positioning the imaging line in the outermost sample chamberof the first radial line; imaging the outermost sample chamber; carryingout image analysis (for example, comprising edge analysis to find theedges of the sample chamber); and rotating the sample holder until themidpoint of the imaged outermost sample chamber is positioned on theoptical axis. This sets the starting position for the imaging to within±50 μm, as measured at the outermost sample chamber.

The optical system (which for example comprises the objective lens andtube lens) may provide a magnification of between 10× and 100×, forexample, and optionally provides a magnification of 10×.

The tube lens may focus the collimated beam coming out of the objectivelens onto the line camera.

The device may be configured to image the or each sample chamber at aplurality of time points. In an AST analysis, for example, the presenceor absence, and/or amount of growth of the pathogen in the samplechamber may be determined at each time point.

The sample holder may comprise focus-verification structures to checkwhether an image captured by the line camera is in focus.

The sample holder may include the samples. The samples may includemicroscopic objects contained in a sample fluid, such as the microscopicobjects discussed below. The fluid may be a liquid with the microscopicobjects in suspension or present on the surfaces of the sample chamberthe sample is contained within. The sample fluid may include clinicalsamples or material derived from clinical samples, wherein the clinicalsamples include, but are not limited to, blood, serum, plasma, bloodfractions, joint fluid, urine, semen, saliva, faeces, cerebrospinalfluid, gastric contents, vaginal secretions, mucus, a tissue biopsysample, tissue homogenates, bone marrow aspirates, bone homogenates,sputum, aspirates, wound exudate, swabs and swab rinsates e.g. anasopharyngeal swab, other bodily fluids and the like. The sample fluidmay include a culture medium and could be a mixture of clinical samplesor material derived from clinical samples with culture medium.

The microscopic objects may include particles (particularlybio-particles), cells (for example mammalian cells such as human cells),micro-organisms such as bacteria, other pathogens such as viruses andfungal pathogens and/or molecules including macromolecules. Themicroscopic objects can include any object that is suitably sized (forexample, having dimensions of less than 500 μm, less than 100 μm, lessthan 50 μm, less than 20 μm, less than 10 μm or less than 1 μm) and canbe detected based on imaging methods, including potentially the use offluorescence of the object or of a fluorescent composition applied tothe object. In some examples, if the objects are translucent objectswithout scattering properties, such as RCP (Rolling Circle Products),the objects may be 10 μm or less in size, perhaps 5 μm in size orsmaller. Such objects may have a largest dimension of 2 μm or less,perhaps 1.5 μm or less, 1 μm or less and in some cases 0.5 μm or less.

As noted above, the samples may include microscopic objects contained ina sample fluid. In an AST analysis, the microscopic objects may includepathogens (for example, bacteria, viruses or fungal pathogens). In sucha case, the pathogens may be present in a sample fluid such as amicrobiological growth medium (for example, cation-adjustedMueller-Hinton broth (CAMHB)), for performing a broth microdilutionassay. The sample chambers may comprise a plurality of antimicrobialagents at a plurality of concentrations.

As also noted above, the samples may include microscopic objects insuspension or present on the surfaces of the sample chamber the sampleis contained within. In some embodiments, substantially all themicroscopic objects are in suspension. In other embodiments, most of themicroscopic objects are in suspension and the remainder are present onthe surfaces of the sample chamber (for example, the bottom surface ofthe sample chamber). In still other embodiments, most of the microscopicobjects are present on the surfaces of the sample chamber (for example,the bottom surface of the sample chamber) and the remainder are insuspension. In other embodiments, substantially all the microscopicobjects are present on the surfaces of the sample chamber (for example,the bottom surface of the sample chamber). The microscopic objects mayinitially be in suspension, but may move over time (for example, bypassive diffusion or under gravity, or using locomotive means such asflagella, in the case of some pathogens) to reach surfaces of the samplechamber (for example, the bottom surface of the sample chamber), and mayattach to those surfaces. Though the sample chambers may be imaged atthe bottom surface of the sample chamber (or just above this surface),in some embodiments, no active steps are taken to get the microscopicobjects to that surface, or to keep them there. That is, the systemoptionally comprises no means for actively getting the microscopicobjects to a surface of the sample chamber (for example, the bottomsurface of the sample chamber), or keeping them at the surface.Alternatively, active steps are taken to get the microscopic objects tothat surface, or to keep them there. That is, the system may comprisemeans for actively getting the microscopic objects to a surface of thesample chamber (for example, the bottom surface of the sample chamber),or keeping them at the surface. For example, the system comprisesagar/agarose at the surface, ligands at the surface, a means for flowingfluid to get the microscopic objects to a surface of the sample chamber(or keep them there) or means for performing electrophoresis,centrifugation, filtration or dielectrophoresis to get the microscopicobjects to a surface of the sample chamber (or keep them there).

In some example embodiments the sample holder is a consumable single-useproduct that can be disposed of after use. This allows for repeated useof the same imaging device without the need for cleaning of the sampleholder, and minimises the risk of contamination of samples.

Optionally, the sample holder is broadly the same shape and size as astandard compact disk (CD). The sample holder may be manufactured usingstandard techniques to make a CD.

In one exemplary sample holder, the sample holder comprises between 1and 600 sample chambers, for example, 50 to 500 sample chambers, and insome examples, 80 to 400 sample chambers, for example 96 chambers, 336chambers or 384 chambers.

In examples having sample chambers following radial lines, there may be12 radial lines of sample chambers, with 8 sample chambers along eachradial line, or 16 radial lines of sample holders, with 6 samplechambers along each radial line, or 24 radial lines of sample holders,with 4 sample chambers along each radial line. Each of the foregoingexamples comprises 96 sample chambers, but there may be more or fewersample chambers. In other examples, there may be 48 radial lines ofsample chambers, with 8 sample chambers along each radial line, or 64radial lines of sample holders, with 6 sample chambers along each radialline, or 94 radial lines of sample holders, with 4 sample chambers alongeach radial line. Each of the foregoing examples comprises 384 samplechambers.

In other configurations, the number of sample chambers along each radialline may not be the same for all radial lines of the sample chamber. Forexample, radial lines of 6 sample chambers and 8 sample chambers mayalternate. In one example, there may be 48 radial lines of samplechambers, with alternating lines of 8 sample chambers and 6 samplechambers.

In examples having sample chambers following coaxial circles (eachcircle lying at a different radius), there may be the same number ofsample chambers on each coaxial circle (for example, there may be 8coaxial circles with 12 sample chambers on each line), but more likely,there will be a different number of sample chambers on each coaxialcircle (for example, an inner coaxial circle may have 4 sample chambers,the next may have 8, the next may have 12, the next may have 16, thenext may have 24 and the outermost may have 32).

In examples having sample chambers following parallel lines, there arefor example 32 parallel lines each having 12 sample chambers, or 16parallel lines each having 24 sample chambers.

Optionally, the sample chambers are broadly rectangular or square incross-section, where the section line is taken in the horizontal plane,parallel to the main (upper and lower) surfaces of the sample holder.Put another way, the bottom surface of the sample chamber is broadlysquare or rectangular.

Each sample chamber has a “length” in the direction along the respectiveradial line, parallel line or coaxial circle, and a “width” in thedirection perpendicular to that line. Here, “length” and “width” arelabels only; no limitation is implied on the relative sizes of thesedimensions. Thus, the width may be greater than, equal to, or smallerthan the “length”. In some embodiments, the sample chambers all have thesame size, shape and aspect ratio (length/width). In other embodiments,one or more of these features may differ between different samplechambers.

Where a single sample chamber is provided, the sample chamber may besubstantially the same size as the sample holder.

The length of the sample chamber(s) may be less than 70 mm, 60 mm, 50mm, 30 mm, 20 mm, 10 mm, 5 mm or 3 mm. The width of the samplechamber(s) may be less than 100 mm, 50 mm, 20 mm, 10 mm, 5 mm, 4 mm or 2mm. The lengths and widths of the sample chambers may for example be 1to 5 mm, for example, 1 to 3.5 mm, or 1.5 to 3 mm.

The sample chambers may have a depth of less than 15 mm, less than 10mm, or less than 5 mm.

The sample chambers may be located along, or distributed along radiallines, concentric circles or parallel lines on the sample holder. Insome embodiments, this means that the sample chambers are all aligned,so that the respective radial line, concentric circle or parallel linepasses through the centre of each sample holder. In some embodiments,the sample chambers generally follow the radial line, concentric circleor parallel line, but are not all aligned with each other. The samplechambers may for example be shifted relative to one another, for examplein an alternating staggered configuration. For example, the centre ofsome or all of the sample chambers may be offset from the radial line,concentric circle or parallel line along which they are distributed. Forexample, a first sample chamber on the radial line, concentric circle orparallel line may be shifted to one side of the radial line, concentriccircle or parallel line, and a neighbouring, second, sample chamber onthe radial line, concentric circle or parallel line may be shifted tothe opposite side of the radial line, concentric circle or parallelline. The next sample chamber may then be aligned with the first, andthe next may be aligned with the second, etc.

As noted above, the line camera line length may be longer than themagnified extent of the sample chambers in the direction perpendicularto the radial line, concentric circle or parallel line. The “extent”depends on both the width of the sample chambers, and how they aredistributed along the radial line, concentric circle or parallel line,i.e. aligned with each other or staggered in some way.

Where the sample holders along one radial line (for example) have equalwidth W and where the sample holders are aligned (such that the radialline passes through the centre of each sample holder), the correspondingouter edges of the sample holders distributed along one radial line arecollinear, and the extent of the sample chambers in the directionperpendicular to the radial line is then the width, W, of the samplechambers. Where the sample chambers are in a staggered configuration,for example where a first sample chamber is offset in one directionperpendicular to the radial line, and another sample chamber is offsetin the opposite direction, if both sample chambers have width W, and thewidths overlap by distance D, the extent of the sample chambersperpendicular to the radial line is 2W-D.

Similar comments apply in respect of sample chambers having staggeredconfigurations along concentric circles or parallel lines.

Focus-checking structures (for example, pyramid-shaped or groove-shapedindentations), may be provided in the sample holder (for example, in abottom layer of the sample holder). Such structures are described inQ-Linea AB's application PCT/EP2017/064715 (WO 2017/216314 A1). Forexample, the focus-checking structures may be provided in the bottom ofeach sample chamber, adjacent each sample chamber (i.e. spaced inwardlyof the outer width of the sample chambers) or may be provided betweenadjacent sample chambers, spaced inwardly of the outer width of thesample chambers. The focal structures may be spaced to appear in every10th line, every 50th line, or every 100th line, captured by the linecamera.

Where the sample holder includes focus checking structures distributedadjacent at least one, a plurality of, or each, sample chamber, spacedoutwardly of the outer width of the sample chambers, the line cameraline length may be longer than the magnified extent of the samplechambers in the direction perpendicular to the radial line, concentriccircle or parallel line, including the focus checking structures.

Where the sample holder includes focus-checking structures (as describedfor example in Q-Linea AB's application PCT/EP2017/064715 (WO2017/216314 A1)), the subsequent image processing may include checkingwhether the images are in focus by checking whether an associated focalstructure is in focus (as described for example in Q-Linea AB'sapplication PCT/EP2017/064711 (WO 2017/216310 A1)). The images acquiredby the device may be analysed using an image analysis algorithm, forexample as described in Q-Linea AB's application PCT/EP2017/064713 (WO2017/216312 A1). The invention is of course not limited to such an imageanalysis; any suitable image analysis method may be used.

The devices and systems described herein may be operated in accordancewith the above described methods, and provide similar advantages. Thus,the device/system may be configured to carry out the method of the firstor second aspects, including any of the preferred/optional/advantageousfeatures of those methods, as described above. The various aspects ofthe invention may comprise any of the optional/preferred/advantageousfeatures of the other aspects of the invention.

Whilst the embodiments discussed above have been discussed in thecontext of bright-field microscopy, fluorescence microscopy could beused. In that case, the microscopic objects can include any object thatis suitably small in size and can be detected based on imaging methodsincluding the use of fluorescence of the object or of a fluorescentcomposition applied to the object.

Moreover, whilst the invention has been described as being particularlyadvantageous in an AST analysis, the invention is of course much moregenerally applicable, for example to drug screening or cell cultureanalyses, also under multiple differing conditions.

Embodiments of the present invention will now be described by referenceto the accompanying figures, in which:

FIGS. 1A and 1B show systems for microscopy-based analysis of samples;

FIG. 2 shows a support for a sample holder which forms part of thesystem of FIG. 1A or FIG. 1B;

FIG. 3 shows a line camera, autofocus system, objective lens and tubelens, arranged as in the system of FIG. 1A;

FIG. 4 shows an exemplary sample holder which could be used in thesystem of FIG. 1A or FIG. 1B;

FIG. 5 illustrates a light beam directed at a focal structure (in thiscase, a pyramid indentation) on an exemplary sample holder and theresultant reflection and refraction of light rays;

FIG. 6A shows the light source incident on an optically active layer ina sample holder, and FIGS. 6B and 6C show modifications to the lightsource to counteract the effect of such an optically active layer;

FIG. 7 shows the movement of the sample holder where the sample holdercomprises sample chambers following radial lines;

FIG. 8 shows the movement of the sample holder where the sample holdercomprises sample chambers following concentric circles (each at adifferent radius); and

FIG. 9 shows the movement of the sample holder where the sample holdercomprises sample chambers following parallel lines.

FIGS. 1A and 1B shows a system for microscopy-based analysis of samples.Each system comprises a device for microscopy-based analysis of samplescomprising a line camera 10, a tracking autofocus system 15, a dichroicmirror 20, an objective lens 25, an illumination light source 30, aband-pass filter 31, a condenser 32, and a tube lens 40. The two systemsin FIGS. 1A and 1B are very similar, the difference being that thelocations of the line camera 10 (and tube lens 40) and autofocus system15 are swapped.

In one example, the line camera 10 is a Linea LA-CM-16K05A (comprising aCMOS digital image sensor) manufactured by Teledyne DALSA, coupled withan XTIUM-CL MX4 frame grabber (not shown), also by Teledyne DALSA. Thecamera array size is 1×16,384 pixels, with each pixel being 3.5 μm×3.5μm. The line width is therefore 3.5 μm, and its length is 57.7 mm. Onlya portion of this length may be used, in practice (for example, fewerthan half of the pixels may be used). The autofocus system 15 comprisesa system from WDI WISE Device Inc., comprising the ATF6 SWIFT digitalautofocus system (with laser wavelength of 785 nm) and an MCZ controllerfor controlling the position of the objective lens 25 in thez-direction. The objective lens 25 is a Nikon CFI Plan-fluor (10×magnification, NA 0.3). The dichroic mirror 20 is a 662 nm edgeBrightLine single-edge imaging-flat dichroic beamsplitter manufacturedby Semrock. The dichroic mirror 20 is held in a holder 21, which isshown in FIG. 3 . The light source 30 comprises an LED light sourceLuxeon LXZ1-PX01 (with central wavelength of about 556-569 nm), acondenser 32, along with a 560/94 nm BrightLine® single-band bandpassfilter 31, manufactured by Semrock. The tube lens 40 is an ITL200 tubelens, from Thorlabs, with a focal length of 200 mm. The condenser 32produces an illuminated area in the plane of the bottom of the samplechamber at the imaging location of approximately 8×8 mm, with thecentral 5×5 mm area having an intensity variation less thanapproximately ±10%. The tube lens 40 focuses the collimated beam comingout of the objective lens 25 onto the line camera 10. The optical systemcomprising the tube lens 40 and the objective lens 25 in this exampleachieves a magnification of 10×.

The system further comprises a sample holder 100 comprising a pluralityof sample chambers 116 (described in greater detail below, withreference to FIG. 4 ). As shown in FIG. 2 , the sample holder 100 isreceived by a support 50 configured to receive the sample holder 100.The support 50 comprises a platform 52 comprising a recessed region 51shaped to conform to the outer dimensions of the sample holder, suchthat, when placed within the recessed region, the sample holder cannotmove laterally.

The platform 52 is provided on linear tracks 56 a, 56 b attached to thesupport, and a motor may be provided to drive the platform in eitherdirection along the tracks. The motor (not shown) may drive movement ofthe platform along the tracks via a rack and pinion arrangement (notshown), for example.

The platform 52 comprises a platform lid 53 which, particularly duringimaging, holds the sample holder 100 in a fixed position with respect tothe vertical axis, i.e. such that the sample holder 100 does not moveupwardly or downwardly.

The platform lid 53 is hingedly connected to the platform, so that itcan pivot upwardly and away from the platform 52 about the hingedconnection. In particular, the platform lid 53 is configured to move inthis way when the platform 52 is translated to an extreme position atone end of the linear tracks 56 a, 56 b (to the far right, as shown inFIG. 2 ). This movement is the result of the platform lid 53 engagingwith a guide rail (not shown), shaped so as to lift the platform lid 53at the extreme position.

The sample holder 100 is loaded from above onto the support 50 (i.e.into the recessed region 51 of the platform 52) at the extreme position.In this position, the sample holder 100 rests within the recessed region51 and is prevented from lateral movement by the recessed region 51. Asthe platform 52 moves from the extreme position, the platform lid 53 isguided down by the guide rail to press down on the sample holder 100, sothat the sample holder 100 is prevented from movement upwardly by thedownward force applied by the platform lid 53. That is, the platform lid53 provides a vertical clamping function. The sample holder 100 isprevented from movement downwardly by being supported by the recessedregion 51.

The support comprises a through-hole 54, below the plane at which thesample holder 100 is supported, which allows a portion of the sampleholder 100 to be imaged by the line camera 10, from below.

In order to bring different radial lines of sample chambers 116 intoline with the line camera 10 for imaging, the support 50 comprises adrive wheel 57 configured to rotate the sample holder 100 (about avertical axis of the sample holder 100). When a sample holder 100 isheld in the support 50, the drive wheel 57 is located adjacent to therim of the sample holder 100, to frictionally engage the rim of thesample holder. The drive wheel 57 is pressed to the rim using a springaction. The drive wheel is driven by a second motor 55, via a drive belt(not shown).

The drive wheel 57 is configured to disengage from the rim of the sampleholder 100 (i.e. the spring action pressing the drive wheel 57 to therim of the sample holder 100 is relaxed) when the platform 52 istranslated to the extreme position at the right-hand end (as shown inFIG. 2 ) of the linear tracks 56 a, 56 b. The drive wheel 57 isconfigured to engage with the rim of the sample holder 100 when theplatform 52 is translated away from the extreme position. The drivewheel is configured to rotate the sample holder 100 at a speed ofapproximately 30° per second.

The support 50 is configured to align the sample holder 100 in aspecific position such that the starting position for the imaging isknown. The support 50 comprises a dedicated detector (for example, aphotodetector, not shown) configured to detect a single alignmentstructure which is present on the sample holder 100 at a distance fromthe centre of the sample holder 100 where no other structures arepresent. This structure defines the absolute position, and then apredetermined offset gives the rotational position of the startingimaging position. The sets the rotational position of the sample holderto within ±500 μm, as measured at the outermost sample chamber. A finepositioning procedure is then done, by translating the platform 52 toposition the imaging line in the outermost sample chamber of the firstradial line, and imaging the sample chamber. Based on image analysis(for example, comprising edge analysis to find the edges of the samplechamber), the sample holder is rotated until the midpoint of the samplechamber is positioned on the optical axis. This sets the startingposition for the imaging to within ±50 μm.

In the use of the device, the sample holder 100 is provided withappropriate samples in sample chambers 116 and images of the samples aregathered using the line camera 10.

Referring to FIG. 1A again, in use, light from the illumination source30 is incident onto the sample holder 100 from above (via the band-passfilter 31 and condenser 32). The light passes through the samplechambers 116 of the sample holder 100, and is collected by the objectivelens 25. After passing through the objective lens 25, the light reflectsfrom the dichroic mirror 20, passes through the tube lens 40, and isthen imaged by the line camera 10.

Similarly, in the system shown in FIG. 1B, in use, light from theillumination source 30 is incident onto the sample holder 100 from above(via the band-pass filter 31 and condenser 32). The light passes throughthe sample chambers 116 of the sample holder 100, and is collected bythe objective lens 25. After passing through the objective lens 25, thelight passes through the dichroic mirror 20, passes through the tubelens 40, and is then imaged by the line camera 10.

The sample holder 100 is moved in a first linear direction (bytranslating the platform 52) in the horizontal plane, such that theimaging line of the line camera 10 successively images different linesperpendicular to the radial line along which the sample chambers 116 aredistributed.

The speed at which the sample holder is translated is, in this example,matched to the imaging rate (line rate) of the line camera, such thatthe resultant image is not distorted. The speed s of the linear movementof the sample holder is given by:

$s = \frac{{pixel}{width} \times {line}{camera}{imaging}{rate}}{magnification}$

Here, the pixel width is 3.5 μm, the line camera imaging rate is 48 kHzand the magnification is 10×. This gives a speed s of 16.8 mm/s. Thisallows imaging of 50 radial lines, each of 50 mm length, within 6minutes (including the time taken for rotation to each new radial line,and data transfers). A sample holder comprising 384 sample chambers canbe fully scanned in 7 minutes. The total analysis time per samplechamber, including movement to the sample chamber, adjusting the focalplane during imaging, and acquiring images within the sample chamber isless than 2 seconds.

Following the completion of the translational movement of the sampleholder 100, the sample holder is rotated by the support 50 (using thedrive wheel 57) in order to bring another radial line of sample chambers116 into alignment with the imaging line of the line camera 10. Thesample holder 100 is then translated in a linear direction in theopposite direction to the first linear direction, to image the secondradial line of sample chambers.

As mentioned, the system comprises an autofocus system 15. The relativepositions of the line camera 10, autofocus system 15, objective lens 25,dichroic mirror 20 (not shown in FIG. 3 , but its holder 21 is shown)and tube lens 40 (in a system similar to that shown in FIG. 1A) areshown in FIG. 3 .

The autofocus system 15 comprises a laser light source (not shown) withwavelength of 785 nm. The laser light 15 a passes through the dichroicmirror 20 and the objective lens 25 (in the opposite direction to thelight gathered by the objective lens 25 from the sample chambers 116),to be incident onto a bottom surface of the sample holder 100. Theautofocus system 15 sets the focal plane at the bottom surface of thesample chambers 116 in the sample holder. The focal plane of the linecamera may be set at a predetermined upward offset therefrom (such thatthe focal plane lies at a plane within the sample chamber 116, above andparallel to the bottom surface of the sample chamber 116), by offsettingthe line camera 10 along the optical axis (by between 0 mm and 20 mm).

The autofocus system 15 can adjust the focal position (if necessary)every 0.15 ms. This allows the autofocus system 15 to recheck the focalposition approximately every 7 lines read by the line camera 10 (whichhas an imaging rate of 48 kHz). If the focal position needs to beadjusted, the autofocus system 15 outputs a signal which causes the lensholder 26 (see FIG. 3 ) to translate the objective lens 25 in order toadjust the focal plane. The lens holder 26 translates the objective lens25 along an axis perpendicular to a plane of the support 50, with aprecision of 1 μm. Movement of the lens holder 26 is driven by a linearactuator (not shown). To image a single sample chamber 116, the linecamera 10 may capture thousands of lines (for example, between 10,000and 15,000), and so the focal plane may be adjusted by the autofocussystem 15 hundreds or thousands of times, across each sample chamber116. Any non-uniformity in the base of the sample chamber 116 cantherefore be accounted for in the imaging process.

As a radial line of sample chambers 116 is imaged by the line camera 10,a composite image comprising the plurality of imaged lines is built up.The composite image obtained by the line camera 10 includes all of thesample chambers 116 along the radial line. This composite image may beprocessed by an image processing algorithm to split the composite intoseparate image areas, each including one sample chamber 116, forexample.

An image analysis system may receive the images taken by the system, andmay carry out further image analysis, for example to determine thepresence, absence, or amount of microscopic objects and/or to determinethe type of microscopic objects (for example, as disclosed in Q-LineaAB's application PCT/EP2017/064713 (WO 2017/216312 A1)).

An exemplary sample holder 100 which is suitable for use with the deviceis now described in greater detail, with reference to FIG. 4 . As seenin this figure, the exemplary sample holder 100 comprises three layers.A first, optically flat, layer 110 forms a base layer. A second layer114 is placed on top of the first layer 110 and is formed with volumes(holes) forming sample chambers 116 for holding sample fluids. Thesample chambers 116 are connected via channels 118. There are multiplechannels 118 each with their own sample chambers 116. The first layer110 closes the bottoms of the sample chambers 116. A third layer 120covers the tops of the sample chambers 116 and the channels 118. Thethird layer 120 includes openings 122 at one end of each of the channels118 to allow for dispensing of sample fluid(s) into each channel 118,and then along the channels 118 to fill all of the sample chambers 116.The third layer 120 also includes vents 124 at the other ends of each ofthe channels 118 to allow for gas to leave the channels 118 as they arefilled with the sample fluid(s). The vents 124 and optionally also theopenings 122 may be covered by a gas permeable membrane. At the end ofeach channel 118 is a reservoir 128 for any excess of the sample fluid.

All of the layers 110, 114, 120 have a central hole 126 that is usedduring loading of the sample holder 100 into the device formicroscopy-based imaging of the samples.

The channels 118 extend outward from the centre of the sample holder 100toward the outer circumference, and they are spaced about along radiallines.

The first layer 110 and the third layer 120 are transparent to light inthe wavelengths used for imaging the samples and are typicallytransparent to visible light. The second layer 114 need not betransparent, although it may be.

In case of use in a fluorescent analysis, the first layer 110, secondlayer 114, and third layer 120 should be non-fluorescent in the relevantwavelength region (for example, 450-700 nm).

Focus-checking structures 112 (for example, pyramid-shaped indentationsor grooves), may be provided in the first layer 110—such afocus-checking structure 112 is shown in FIG. 5 . Such structures aredescribed in Q-Linea AB's application PCT/EP2017/064715 (WO 2017/216314A1)). The focus-checking structures may be provided in the bottom ofeach sample chamber 116, at the end of each channel 118, adjacent eachsample chamber 116 or adjacent each channel 118. In another arrangement,each channel 118 may have a plurality of associated focal structures 112spaced at set distances from the centre of the sample holder 110, suchthat the focal structures 112 lie along concentric circles centred onthe centre of the sample holder 100. The focal structures 112 may beprovided between adjacent sample chambers 116, spaced inwardly of theouter width of the sample chambers 116. The focal structures 112 may bespaced to appear in every 10th line, every 50th line, or every 100thline, captured by the line camera.

As shown in FIG. 5 , a collimated light beam perpendicular to the flatsurface of the first layer 110 gives rise to total internal reflectionon the sidewalls of the focus checking structure 112. In the case of aless than perfectly collimated beam, the reflection may not be total,but it is still sufficient for contrast detection as detailed below. Asa result of the (total) internal reflection, when viewed from the top,the majority of the area of the focus-checking structure 112 appearsdark. If the line camera 10 is focused exactly on the base of thefocus-checking structure 112, where the sidewalls meet and form thepoint of the pyramid indentation, then a bright spot appears. Thecontrast between this bright spot and the darker area of the surroundingpart of the pyramid changes rapidly with changing focal plane.

As explained above, as the line of sample chambers 116 is imaged by theline camera 10, a composite image comprising the plurality of imagedlines is built up. The composite image obtained by the line camera 10includes all of the sample chambers 116 and focal structures 112 alongthe channel 118. This composite image may be processed by an imageprocessing algorithm to split the composite into separate image areas,each including a sample chamber and at least one focal structure 112. Inone example, the focal structure 112 associated with a given samplechamber 116 comprises two pyramid indentations at each end of the samplechamber 116. In another example, there is a focal structure 112comprising four pyramid indentations 30 at the end of each samplechamber 116. In each case the geometry (i.e. layout of the pyramidindentations) may be the same, but the subsequent association of a focalstructure 112 with a sample chamber 116 in the imaging processing isdifferent.

An image analysis system may check the images to determine if they arein focus by identifying the focal structures 112 and checking whether ornot they are in focus (as described for example in Q-Linea AB'sapplication PCT/EP2017/064711 (WO 2017/216310 A1)). If any of the imagesare not in focus then an indication can be given to the user and/orremedial action can be taken.

Referring to FIGS. 6A to 6C, in some embodiments, the third layer 120 ofthe sample holder 100 may be optically active, and causes non-uniformityin the light incident onto the sample chambers. In particular, theoptically active layer may comprise structures that refract or blocklight so that the illumination intensity as perceived over the imagedareas is not even, but shows variations dependent on the shape of thethird layer 120. Such variations may be detrimental to the image, andsubsequent image processing. To counteract this, a diffuser 60 may bepositioned between the illumination source 30 and the third layer 120 ofthe sample holder 100 (as shown in FIG. 6B). The diffuser may be anoptical diffuser which diffuses the light evenly, or it may be anengineered diffuser comprising an engineered surface having structuresdesigned to cancel out the light intensity variations caused by theoptically active part of the sample holder. Alternatively, a pluralityof light sources 30′ may be provided (as shown in FIG. 6C), positionedto provide different path lengths for illumination of the samplechambers. The diffuser 60 or plurality of light sources 30′ act toprovide a more even illumination to the sample chambers.

In the foregoing description, the sample holder 100 comprises samplechambers aligned along radial lines. The movement of such a sampleholder (in order to image the sample chambers) is shown in more detailin FIG. 7 .

The sample chambers (not shown in FIG. 7 ) on the sample holder aredistributed along strips S₁, S₂ and S₃, broadly following radial linesL₁, L₂ and L₃. The strips are defined by the line camera active linelength W (number of pixels used×pixel length—the line camera active linelength may be smaller than the maximum line length, because the numberof pixels used may be fewer than the total number of pixels) divided bythe magnification M, and the path the imaging line takes over the sampleholder (as a result of movement of the sample holder relative to theimaging line). There may be a plurality of sample chambers along eachstrip S₁, S₂, S₃ or there may be one long sample chamber per strip.Alternatively, there may be just one sample chamber, imaged in aplurality of strips S₁, S₂, S₃. In FIG. 7 , the strips are shown as notoverlapping, but in other embodiments they may overlap (or abut, withoutsignificant overlap).

In FIG. 7 , the support translates the sample holder linearly in a firstdirection T₁ (by translating the platform 52), which moves the sampleholder such that the first strip S₁ moves across the imaging line from aradially outward position towards the centre of the sample holder. Thefirst strip S₁ is imaged as the strip is translated over the imagingline. When sample holder is positioned such that the imaging line is atthe radially inward end of the first strip S₁, the sample holder isrotated using the drive wheel 57 (in direction R₁, which in this case isclockwise) to bring the radially inward imaging position of the secondstrip S₂ into alignment with the imaging line of the line camera. Thesupport then translates the sample holder linearly in a second directionT₂, (the opposite direction from T₁) by translating the platform 52which in this case moves the second strip S₂ across the imaging linefrom a radially inward position towards the outer edge of the sampleholder. The second strip S₂ is thus imaged. When the sample holder ispositioned such that the imaging line is positioned at the radiallyoutward end of the first strip S₂, the sample holder is rotated (indirection R₁, using the drive wheel 57) to bring the radially outwardimaging position on the third strip S₃ into position above the imagingline. The support then translates the sample holder linearly in thefirst direction T₁ by translating the platform 52, for imaging of thethird strip S₃.

In alternative embodiments, the sample holder comprises sample chambersaligned along concentric circles (positioned at different radii). Themovement of the sample holder in order to image the sample chambers isshown in more detail in FIG. 8 .

The sample chambers (not shown in FIG. 8 ) on the sample holder aredistributed along strips S₁′ and S₂′, broadly following concentriccircles lines L₁′ and L₂′. The strips are defined by the line cameraactive line length W (number of pixels used×pixel length) divided by themagnification, and the path the imaging line takes over the sampleholder (as a result of movement of the sample holder relative to theimaging line). There may be a plurality of sample chambers along eachstrip S₁′, S₂′ or there may be one long sample chamber per strip.Alternatively, there may be just one sample chamber on the sampleholder, imaged in a plurality of strips S₁′, S₂. In FIG. 8 , the stripsare shown as not overlapping, but in other embodiments they may overlap(or abut, without any significant overlap).

In FIG. 8 , the support rotates the sample holder in a first directionR₁′ (which in this case is clockwise) using the drive wheel 57, whichmoves the sample holder such that the first strip S₁′ moves across theimaging line. The first strip S₁′ is imaged as the strip is moved overthe imaging line. When the sample holder is positioned such that theimaging line returns to the initial position on the first strip S₁′(i.e. when the sample holder has been rotated by 360°), the sampleholder is translated linearly in direction T₁′ by translating theplatform 52, which in this case causes the imaging line to be positionedat a radially outward position, such that a position of the second stripS₂′ is brought into alignment with the imaging line of the line camera.The support then rotates the sample holder using the drive wheel in thefirst direction R₁′ (although the support could instead rotate thesample holder in the opposite direction, i.e. anti-clockwise), whichmoves the sample holder such that the second strip S₂′ moves across theimaging line. The first strip S₂′ is imaged as the strip is moved overthe imaging line.

In alternative embodiments, the sample holder comprises sample chambersaligned along parallel lines. The movement of the sample holder in orderto image the sample chambers is shown in more detail in FIG. 9 .

The sample chambers (not shown in FIG. 9 ) on the sample holder aredistributed along strips S₁″, S₂″ and S₃″, broadly following parallellines L₁″, L₂″ and L₃″. The strips are defined by the line camera activeline length W (number of pixels used×pixel length) divided by themagnification M, and the path the imaging line takes over the sampleholder (as a result of movement of the sample holder relative to theimaging line). There may be a plurality of sample chambers along eachstrip S₁″, S₂″, S₃″ or there may be one long sample chamber per strip.Alternatively, there may be just one sample chamber on the sampleholder, imaged in a plurality of strips S₁″, S₂″, S₃″. In FIG. 9 , thestrips are shown as not overlapping, but in other embodiments they mayoverlap (or abut, without any significant overlap).

In FIG. 9 , the support translates the sample holder linearly in a firstdirection T₁ by translating the platform 52, which moves the sampleholder such that the first strip S₁ moves across the imaging line. Thefirst strip S₁ is imaged as the strip is translated over the imagingline. When the sample holder is positioned such that the imaging line isat a far end of the first strip S₁, the support then translates thesample holder by translating the platform 52 linearly in a seconddirection T₂″, (at an angle to T₁″, and in this case perpendicular toT₁″) to bring an end of the second strip S₂″ into alignment with theimaging line of the line camera. The support translates the sampleholder linearly in a third direction T₃″ (opposite to T₁″) bytranslating the platform 52, which moves the sample holder such that thefirst strip S₂ moves across the imaging line. When the sample holder ispositioned such that the imaging line is at a far end of the secondstrip S₂″, the support then translates the sample holder linearly in thesecond direction T₂ again (by translating the platform 52), to bring anend of the third strip S₃″ into alignment with the imaging line of theline camera. The support then translates the sample holder linearly inthe first direction T₁″ again (by translating the platform 52), whichmoves the sample holder such that the third strip S₃″ moves across theimaging line.

Where the sample holder comprises sample chambers aligned along parallellines, there is no need for the drive wheel 57 in the system describedin FIGS. 1A and 1B, 2 and 3 . In that case, the platform 52 may beprovided on additional linear tracks attached to the support, which areat an angle to, for example perpendicular to, the tracks 56 a, 56 bshown in FIG. 2 . Movement along these tracks may be driven by the samemotor which drives the motion along the first linear tracks 56 a, 56 b,or by a different motor.

1. An imaging device for microscopy-based imaging of samples comprising:a line camera; a support, configured to receive a sample holder; anobjective lens received by a lens holder, wherein the lens holder isoperable to move the objective lens along an optical axis; and anautofocus system, wherein the support is configured to move the sampleholder in a first direction relative to an imaging line of the linecamera to capture an image of a first strip of the sample holder,wherein the autofocus system is configured to determine a focal plane,and is configured to output a signal which causes the lens holder totranslate the objective lens in order to adjust the focal plane, duringmovement of the sample holder in the first direction by the support, andwherein the support is configured to move the sample holder in a seconddirection to align the imaging line of the line camera with a positionon a second strip of the sample holder, wherein the first direction is astraight line, and the second direction is a rotational direction. 2.The imaging device according to claim 1, wherein the device comprises aframe grabber configured to create an area image from a plurality ofserially captured line images captured by the line camera.
 3. Theimaging device according to claim 1, wherein the autofocus system is atracking autofocus system, and is configured to adjust the focal planeas the sample holder moves, and optionally, the autofocus system isconfigured to adjust the focal position at least every 0.5 ms.
 4. Theimaging device according to claim 1, wherein the autofocus systemcomprises an autofocus system light source, wherein the autofocus lightsource is an additional light source that is different from anillumination source and has a different wavelength from the wavelengthof the illumination source.
 5. The imaging device according to claim 1,wherein the light from the autofocus system light source is configuredto pass through the objective lens, to be incident onto a bottom surfaceof the sample holder.
 6. The imaging device according to claim 1,wherein a focal plane of the line camera is set at the same plane as thefocal plane determined by the autofocus system.
 7. An imaging deviceaccording to claim 1, wherein the line camera is mounted with a slightoffset in the direction of the optical axis in order to place the focalplane of the line camera at a different level than the focal plane forthe autofocus system, wherein optionally the line camera is offset bygreater than 0 mm and less than 20 mm, for example by 15 mm, 10 mm, 5mm, 2 mm or 1 mm.
 8. An imaging device according to claim 1, wherein thedevice comprises a dichroic mirror, and optionally wherein the dichroicmirror is configured to be broadly transparent to light from anillumination source but to reflect light from the autofocus system, oris configured to be broadly transparent to light from the autofocussystem but to reflect the light from an illumination source.
 9. Animaging device according to claim 1, wherein the optical axis alongwhich the lens holder is configured to move the objective lens isvertical and the objective lens is configured to be moved substantiallyvertically upwardly or downwardly.
 10. An imaging device according toclaim 1, wherein the line camera is configured to image the sampleholder from below a plane at which the support is arranged to hold thesample holder.
 11. An imaging device according to claim 1, wherein theline camera is mounted in a fixed position such that it does not move.12. An imaging device according to claim 1, wherein the imaging devicecomprises an illumination source, wherein the illumination source isoptionally monochromatic, or a narrow-band source, and/or wherein theillumination source is provided at a position above the support toilluminate the sample holder from above.
 13. An imaging device accordingto claim 12, wherein the device comprises a condenser, for shaping thelight beam emitted from the illumination source to provide substantiallyhomogenous illumination at the position where the sample holder isimaged by the line camera.
 14. An imaging device according to claim 1,wherein the device comprises a tube lens, for focusing a collimated beamcoming out of the objective lens onto the line camera.
 15. An imagingdevice according to claim 12, wherein the device comprises a narrow-bandsingle band bandpass filter, wherein the central wavelength of the bandis at approximately the central wavelength of the illumination source,and/or wherein the bandwidth of the bandpass filter is less than 200 nm,and is optionally approximately 100 nm, or less.
 16. An imaging deviceaccording to claim 12, wherein the illumination source comprises aplurality of light sources; and/or wherein a diffuser is positionedbetween the illumination source and the sample holder.
 17. An imagingdevice according to claim 1, wherein the support comprises a platformcomprising a recessed region shaped to conform to the outer dimensionsof the sample holder.
 18. An imaging device according to claim 17,wherein the platform is provided on linear tracks attached to thesupport, allowing the platform to be linearly translated.
 19. An imagingdevice according to claim 17, wherein the platform comprises a platformlid which is hingedly connected to the platform, and optionally whereinthe platform lid is caused to move upwardly and downwardly, for exampleby engagement with a guide rail.
 20. An imaging device according toclaim 1, wherein the support comprises a drive wheel configured torotate the sample holder about a vertical axis of the sample holder,wherein optionally, the drive wheel is located adjacent to the rim ofthe sample holder, to frictionally engage a rim of the sample holder,and/or wherein the drive wheel is configured so as to disengage from arim of the sample holder to allow the sample holder to be removed fromthe support.
 21. An imaging device according to claim 1, wherein thesupport comprises a detector, optionally a photodetector, configured todetect a single alignment structure which is present on the sampleholder at a distance from the centre of the sample holder where no otherstructures are present, in order to determine an absolute rotationalposition of the sample holder, and optionally wherein the device isconfigured to carry out an adjustment to the rotational position by:positioning the imaging line in the outermost sample chamber of thefirst radial line; imaging the outermost sample chamber; carrying outimage analysis, for example, comprising edge analysis to find the edgesof the sample chamber; and rotating the sample holder until the midpointof the imaged sample chamber is positioned on the optical axis.
 22. Asystem for microscopy-based imaging of samples comprising the imagingdevice as claimed in claim 1, and a sample holder.
 23. A system asclaimed in claim 22, wherein the sample holder comprises a single samplechamber, or wherein the sample holder comprises a plurality of sampleholders, for example up to 600 sample chambers, for example, 50 to 500sample chambers, for example 80 to 400 sample chambers, for example 96chambers, 336 chambers or 384 chambers.
 24. A system as claimed in claim22, wherein the sample holder comprises a plurality of sample chamberslocated along radial lines of the sample holder, wherein a single samplechamber is located along each radial line, or a plurality of samplechambers are located along each radial line.
 25. A system as claimed inclaim 22, wherein the sample holder comprises a plurality of samplechambers located along a plurality of concentric circles havingdifferent radii on the sample holder, wherein a single sample chamber islocated along each coaxial circle, or a plurality of sample chambers arelocated along each coaxial circle.
 26. A system as claimed in claim 22,wherein the sample holder comprises a plurality of sample chamberslocated along a plurality of parallel lines, wherein a single samplechamber is located along each parallel line, or a plurality of samplechambers are located along each parallel line.
 27. A system as claimedin claim 22, wherein the sample holder comprises focus-checkingstructures, which are optionally pyramid-shaped or groove-shapedindentations, wherein optionally the focal structures are spaced toappear in every 10th line, every 50th line, or every 100th line,captured by the line camera.
 28. A system as claimed in claim 22,wherein the sample chambers include a plurality of antimicrobial agents,each at a plurality of concentrations, for performing an AST analysis.29. A system as claimed in claim 22, wherein the sample holder comprisessamples, which include microscopic objects contained in a sample fluid.30. A system as claimed in claim 29, wherein the samples includepathogens present in a microbiological growth medium for performing abroth microdilution assay.